Project 3: Functional Neuro circuitry of Energy Balance (P.I. Bradford B/ Lowell) Neuroanatomical mapping studies have identified neural pathways which are likely to be important in regulating food intake and energy expenditure. However, these anatomical studies are unable to establish the functional importance of these connections. The present proposal attempts to address this issue by using transgenic and conditional gene targeting methods to assess the functional significance of leptin receptors and MC4-Rs on anatomically restricted subclasses of neurons. The following specific aims are proposed: Specific Aim #1: To assess the functional importance of leptin receptors on POMC and AgRP expressing neurons. Mice lacking long-form leptin receptors (db/db mice) are obese. Thus, long-form leptin receptors play a critical role in controlling energy balance. Based upon many pieces of evidence outlined within this grant, we hypothesize that long-form leptin receptors on AgRP-positive and/or POMC-positive neurons in the arcuate nucleus mediate the anti-obesity and neuroendocrine actions of leptin. To test this hypothesis, we will generate transgenic mice, on the db/db background, which express long-form leptin receptors in either AgRP- positive neurons, POMC-positive neurons, or both. This will be accomplished by constructing transgenes in which either AgRP or POMC promoter elements drive expression of long-form leptin receptors. Specific Aim #2: To assess the functional importance of melanocortin 4 receptors (MC4-Rs) on neurons in the lateral hypothalamic area (LHA) and the paraventricular hypothalamic nucleus (PVN). Multiple lines of evidence indicate that MC4-Rs play a critical role in controlling body "anti-obesity" effects of MC4-Rs. Based upon the projects of alpha-MSH and AgRP- positive cell bodies in the arcuate nucleus, to cell groups thought to be important in the LHA and PVN, we hypothesize that MC4-Rs on neurons in the LHA and/or PVN mediate the anti-obesity effects of MC-4 agonists. Based upon the projections of alpha-MSH and AgRP-positive cell bodies in the neurons in the LHA and/or PVN mediate the anti-obesity effects of MC4-agonists. To test this hypothesis, we will use homologous recombination in embryonic stem cells to generate a lox-modified, null allele of the MC4-R gene. The null allele will be designed so that its function an be reactivated by the action of cre-recombinase. We will inject adeno-associated virus (AAV) expressing cre-recombinase into either the LHA or PVN of homozygous null MC4-R mice to reactive the MC4-R gene in these key anatomic sites.